The hepatic progenitor HP14-19 cell line expressing the HSV-tk suicide gene and SV40T immortalized gene was constructed previously (11,12). Cells were cultured in complete Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO₂.

A ~1,300 bp fragment of the full-length CD gene from Escherichia coli JM107 genomic DNA was amplified using the polymerase chain reaction (PCR). A BamHI restriction site was added to the 5′-end of the forward primer, and a SalI restriction site was added to the 5′-end of the reverse primer. The primer sequences were as follows: 5′-CGAGGATCCATGTCGAATAACGCTTTAC-3′ (forward) and 5′-GCTGTCGACTCAACGTTTGTAATCGATG-3′ (reverse). The target CD gene fragment was amplified by touch-down PCR with a reaction system consisting of 4 µg DNA template, 2 µl 330 µmol/l upstream and downstream primers, 25 µl 2X high-fidelity master mix (Novoprotein Technology Corporation, Shanghai, China), and double-distilled water to 50 µl, and the following thermocycling conditions: 95°C for 3 min, followed by 10 cycles of 92°C for 20 sec, 65°C for 20 sec and 72°C for 120 sec (with 1°C decrease per cycle), and then 25 cycles of 92°C for 20 sec, 55°C for 20 sec and 72°C for 120 sec, and finally 72°C for 3 min. Following detection by 1% agarose gel electrophoresis, the PCR products were digested with BamHI/SalI and then directionally cloned into the BglII/SalI-digested pSEB-C3F retroviral vector (Molecular Oncology Laboratory, The University of Chicago Medical Center, Chicago, IL, USA). The constructed pSEB-CD plasmid was selected by colony PCR, and confirmed by full-length PCR, double enzyme digestion (EcoRI and SalI) and sequencing (Huada Gene, Inc., Shenzhen, China). All enzymes were purchased from Takara Biotechnology Co., Ltd., Dalian, China.

The pSEB-CD plasmid expressing a blasticidin S (BSD) selection marker and pCLAmpho mammalian expression vector (Molecular Oncology Laboratory, The University of Chicago Medical Center) were co-transfected into 293 cells to package the retrovirus at 37°C and 5% CO₂ for 7 days. HP14-19 cells were then seeded into a T-25 flask and infected with retrovirus at 37°C and 5% CO₂ for 7 days; they were then selected in the presence of 3 µg/ml BSD (Invitrogen; Thermo Fisher Scientific, Inc.) for 14 days.

Total RNA from HP14-19 cells and HP-14-19-CD cells was extracted using an RNA Extraction kit (BioTeke Corporation, Beijing, China) at the indicated times and reverse-transcribed into cDNA using Superscript II reverse transcriptase (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The cDNA samples were diluted 5–10-fold and subjected to PCR amplifi-cation with a reaction system consisting of 2 µg DNA template, 1 µl 330 µmol/l upstream and downstream primers, 7.5 µl 2X Taq master mix (Novoprotein Technology Corporation), and double-distilled water to 15 µl. PCR primers were designed using the Primer3 program (Table I). A touch-down PCR protocol was performed with the following thermocycling conditions: 95°C for 3 min, then 10 cycles of 92°C for 20 sec, 65°C for 20 sec and 72°C for 20 sec (with 1°C decrease per cycle), followed by 30 cycles of 92°C for 20 sec, 55°C for 20 sec and 72°C for 20 sec, and finally 72°C for 3 min. The PCR products were electrophoresed on a 1.5% agarose gel. All samples were normalized to the expression level of GAPDH.

Cells were lysed in radioimmunopre-cipitation assay buffer with phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China). The protein concentrations were determined with a Bicinchoninic Acid Protein assay kit (Beyotime Institute of Biotechnology). Total protein (20 µg/group) was separated by SDS-PAGE (SV40T and HSV-tk protein were separated on a 10% gel; CD and β-actin proteins were separated on a 15% gel). Beyotime Institute of Biotechnology) and then transferred onto a poly-vinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk (Beyotime Institute of Biotechnology) in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h and then incubated at 4°C overnight with primary antibodies against SV40T (1:200; cat. no. sc-148; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), HSV-tk (1:200; cat. no. sc-28037; Santa Cruz Biotechnology, Inc.), CD (1:1,000; cat. no. ab35251; Abcam, Cambridge, MA, USA) and β-actin (1:200; cat. no. sc-47718; Santa Cruz Biotechnology, Inc.). Following washing with TBST, the membrane was incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (goat anti-mouse, 1:1,000, cat. no. sc-2302 and goat anti-rabbit, 1:1,000, cat no. sc-2004; both from Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Finally, the protein expression levels were determined using an enhanced chemiluminescent substrate (Nanjing Kaiji Biotechnology Co., Nanjing, China) and exposed under the Syngene G-Box Imaging system (Syngene Europe, Cambridge, UK).

HP14-19-CD cells were infected with adenovirus Ad-Cre (12) to remove the HSV-tk gene, then cultured in complete Dulbecco's modified Eagle's medium with 10% FBS containing various concentrations (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 µg/ml) of GCV (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). HP14-19 and HP14-19-CD cells were treated with various concentrations (0, 10, 20, 40 and 80 µg/ml) of 5-FC (Sigma-Aldrich; Merck KGaA). HP14-19-CD cells which were infected with Ad-green fluorescent protein served as control group. Crystal violet staining was performed in order to assess cell viability. Cells were incubated in 24-well plates at 2.0×10⁴ cells/well and analyzed after 1 week. Briefly, the culture medium was removed and the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, then stained with 0.05% crystal violet for 30 min. Following washing with tap water, cells were dried on filter paper. The blue dye was dissolved in 500 µl methanol and determined at an excitation wavelength of 540 nm using a Multimode Microplate Reader (Thermo Fisher Scientific, Inc.). Three independent experiments were performed in triplicate. The mean and standard deviation were calculated.

The use and care of animals was approved by the Institutional Animal Care and Use Committee of The Children's Hospital of Chongqing Medical University (Chongqing, China). Nude mice (3 males and 3 females, 5–6 weeks of age, 22–23 g) were purchased from Tengxin Institute of Biotechnology (Chongqing, China). The animals were kept at room temperature between 22 and 26°C with 40–60% relative humidity and a 12-h light/12-h dark cycle with free access to food and water. HP14-19 and HP14-19-CD cells were labelled with adenovirus (Ad)-firefly luciferase and then subcutaneously inoculated in the front and rear notum on the left and/or right side of the 6 nude mice (1×10⁶ cells per injection). 5-FC (300 mg/kg per day) was intragastrically administered to the nude mice in vivo every 2 days following cell transplantation. At 0, 5 and 10 days after implantation, mice were intraperitoneally injected with 0.1 ml D-luciferin (Gold Biotechnology, Inc., St Louis, MO, USA) at 2 mg/ml, and visualized using the IVIS-200 optical in vivo imaging system (Xenogen Corporation, Alameda, CA, USA) to dynamically observe the luciferase signals and determine the survival rate of cells.

A total of 21 nude mice (all male, 5–6 weeks of age, 22–23 g) were purchased from Tengxin Institute of Biotechnology. The animals were kept at room temperature between 22 and 26°C with 40–60% relative humidity and a 12-h light/12-h dark cycle, and were randomly divided into a normal group (n=3), a 2% carbon tetrachloride (CCl₄) group (n=9) and a CCl₄+cells group (n=9). A total of 18 nude mice were used to construct an acute liver injury model established via 2% CCl₄ gavage. Considering the large amount of haemorrhagia during the procedure and a typically low survival rate following portal vein injection, it was elected to transplant cells via the splenic vein (13). Cells were pre-labeled with Hoechst 33342 (Beyotime Institute of Biotechnology) 24 h after liver injury (14,15). The liver index (liver wet weight/body weight ×100%), serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in each group were detected using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at the indicated time points.

Following sacrifice of the mice, liver tissue specimens were obtained and fixed in 4% para-formaldehyde, embedded in paraffin following dehydration, and serially cut into 5-µm sections. Serial sections were then stained with 0.2% hematoxylin and 0.5% eosin (H&E) at room temperature, and images were captured at ×200 magnification under a light microscope (Nikon Corporation, Tokyo, Japan).

All data are presented as the mean ± standard deviation and were analyzed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). Statistical analysis was performed using the two-tailed Student's t-test to determine significant differences between two groups, and one-way analysis of variance followed by Tukey's honest significant difference post hoc test was performed to determined significant differences among at least three groups. P<0.05 was considered to indicate a statistically significant difference.

A ~1,300 bp fragment of the full-length CD gene from E. coli JM107 genomic DNA was amplified using PCR. This ~1,300 bp DNA fragment could be removed from the constructed pSEB-CD plasmid by digestion with SalI and BamHI restriction enzymes. In addition, the target genes were confirmed to be correctly cloned in the pSEB-C3F vector by gene sequencing and matched the CD sequence in GenBank^® (www.ncbi.nlm.nih.gov/genbank) (Fig. 2). The HP14-19 cell line contains the hygromycin-resistant/HSV-tk fusion gene and immortalized SV40T, which can be removed by Cre/LoxP recombination. As the pSEB-C3F vector contained the BSD resistance gene, following infection with the pSEB-CD-produced retrovirus, the cells also acquired the BSD resistance gene and thus were able to survive in the presence of BSD (Fig. 3A). HP14-19 and HP14-19-CD cells expressed the SV40T and HSV-tk genes; however, only HP14-19-CD cells stably exhibited CD mRNA and protein expression (Fig. 3B). Therefore, the procedure of the present study successfully established long-term cell culture of hepatic progenitor cells containing immortalizing SV40T and double suicide genes; the HSV-tk and stable CD genes could also be subsequently knocked out.

As the HP14-19-CD cells contained the HSV-tk suicide gene, cell death was dose-dependently induced by the addition of GCV. HSV-tk expression was controlled by Cre-LoxP site-specific recombination. Ad-mediated Cre expression was able to inhibit the expression of HSV-tk, thus Ad-Cre-infected cells were able to survive, even in the presence of high-dose GCV medium (P<0.05; Fig. 4A). The CD suicide gene codes for cytosine deaminase, which converts the non-toxic compound 5-FC into 5-fluorouracil to induce cell death. As presented in Fig. 3B, when exposed to various doses of 5-FC, the rate of HP14-19-CD cell death was significantly increased compared with that of HP14-19 cells (P<0.05; Fig. 4B).

The efficiency of Cre-LoxP-mediated recombination was not 100%, thus Ad-Cre-infected HP14-19-CD cells still expressed SV40T and HSV-tk mRNA and protein at a low level. However, using the negative selection with GCV, all cells expressing the SV40T immortalization and HSV-tk genes were removed, and the selected cells still expressed the CD gene and protein (Fig. 5A and B).

Next, the effect of the CD gene on cell suicide was investigated in vivo. HP14-19 and HP14-19-CD cells were labelled with Ad-firefly luciferase and then subcutaneously inoculated into nude mice. Optical in vivo imaging was employed to observe luciferase signaling in surviving cells. As presented in Fig. 6, the original luciferase signal of HP14-19 and HP14-19-CD cells was markedly detectable on day 0; the luciferase signal of HP14-19 cells decreased slowly and remained easily detectable on day 10. By contrast, the luciferase signal exhibited by HP14-19-CD cells was weaker compared with that of HP14-19 cells, and was almost impossible to detect on day 10. These results demonstrated that the immortalization of HP14-19-CD cells could be successfully altered, while maintaining its biosafety in vivo with CD gene expression.

The recovery efficiency of HP14-19-CD cells in vivo was next investigated. First, an ALF model was successfully established using 2% CCl₄ gavage. Compared with normal nude mice, the liver index of the ALF model was significantly increased (P<0.05), and the levels of ALT and AST also increased >10-fold (P<0.05). H&E staining revealed that there was hepatocyte degeneration in the ALF liver tissues, as well as hepatocyte steatosis, vacuolar degeneration, focal necrosis, hepatic cord disorder, and nuclear condensation or disappearance (Fig. 7A). Hoechst 33342-pre-labelled cells were injected into the splenic vein, and the majority of cells were distributed around the portal vein in the hepatic portal area on day 1 post-transplantation. After 14 days, the majority of the cells had survived and a proportion of cells had migrated into the liver parenchyma (Fig. 7B). The liver has a good ability to self-repair, thus, when the model mice no longer received CCl₄ stimulation, gradually the liver index, ALT/AST levels and hepatic pathological changes recovered to a certain extent. The liver index of the cell+CCl₄ group was consistently lower compared with that of the CCl₄ group, with a significant difference observed on day 3; however, no significant differences were identified following day 7 (Fig. 7C). The ALT and AST levels of the cell+CCl₄ group were significantly decreased compared with the CCl₄ group at each time point (Fig. 7D). The H&E staining results presented in Fig. 7E revealed that, when compared with the CCl₄ model group, the transplanted cells markedly repaired the liver pathological structure. Damaged hepatocytes were markedly repaired on the third day following cell injections, whereas the structure of the hepatic cords had almost completely recovered after 14 days. Therefore, these results indicated that HP14-19-CD cell transplantation may improve hepatic recovery.